Apparatus for detecting biopolymers and cartridge

ABSTRACT

Provided is an apparatus for detecting biopolymers (DNA) capable of total analysis including non-reacted samples without complicated operations such as washing. 
     A DNA probe is fixed to one of electrodes and direct current voltage is placed between the electrodes, so that it becomes possible to separate complementary strand sample DNA and non-complementary strand sample DNA. By analyzing from a ratio in the whole reaction system, it is possible to obtain clearer results. Further, by using electrophoresis by gel together, it is possible to separate reacted samples and non-reacted samples to perform measurements therefor in the same reaction field.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. Ser. No. 10/001,012 filed Nov.30, 2001 now U.S. Pat. No. 6,875,603. Priority is claimed based on U.S.Ser. No. 10/001,012 filed Nov. 30, 2001, which claims priority toJapanese Patent Application No. 2000-364370 filed on Nov. 30, 2000.

PRIORITY INFORMATION

This application claims priority to Japanese Application Serial No.364370/2000, filed Nov. 30, 2000.

BACKGROUND OF THE INVENTION

The present invention relates to an apparatus for detecting biopolymerscapable of detecting the presence of biopolymers such as DNA, RNA andprotein in a sample and measuring an existing amount or a concentrationthereof, and to a cartridge used for the detection.

As technologies for detecting DNA, such technology has been generallyused, in which DNA is modified with a radioactive material, afluorescence dyestuff or the like by use of technologies of RI(radioactive isotope), fluorescence or the like and excited by astimulus from the outside for detection of response by luminescence.Also an electric charge detecting method for electrochemicallydetermining DNA based on an oxidation-reduction potential by use of anintercalating agent, which is specifically bonded to a duplex of DNA,has been devised. Further, there is a method of using a surface plasmonresonance phenomenon as a method without modification and the like. Withrespect to a method of fixing DNA to an electrode, there is a method ofutilizing an action that a monolayer of free thiol radicals located onthe end of DNA is self-organized on the surface of gold using a thiolmodified DNA probe.

In conventional DNA detecting technologies, methods of using RI orfluorescence have been needed to modify DNA.

SUMMARY OF THE INVENTION

An apparatus for detecting biopolymers in accordance with the presentinvention includes: a voltage supply unit for placing electric voltagebetween two electrodes of a cartridge which stores biopolymers betweenthe electrodes; a holding unit for holding the cartridge; an irradiationunit for irradiating light onto the cartridge held by the holding unit;and a light receiving unit for receiving the light irradiated by theirradiation unit onto the cartridge held by the holding unit.

The voltage supply unit can selectively supply alternating currentvoltage and direct current voltage so that biopolymers can be attractedto one electrode or both electrodes.

The holding unit can two-dimensionally move the cartridge on a planeperpendicular to an optical axis of the light irradiated by theirradiation unit so that the presence of a biopolymer on each locationin the cartridge can be detected.

Since the irradiation unit can irradiate light having a specified singlewavelength, sensitivity for detecting can be improved.

The apparatus for detecting biopolymers further includes an arithmeticunit for calculating an existing amount, a base length, a concentration,a hybridization ratio and a hybridization amount of a biopolymer from aquantity of light received by the light receiving unit so that variouskinds of feature amounts for the biopolymer can be determined.

The apparatus for detecting biopolymers further includes a heater whichapplies heat to the electrodes of the cartridge for disassociatingbiopolymers hybridized in the cartridge to single strands, so that eachpresence of a complementary strand biopolymer and a non-complementarystrand biopolymer can be detected.

Also, a cartridge in accordance with the present invention includes: apillar-shape base unit capable of accommodating a biopolymer solution,the base unit having a first electrode on the inside of a bottom face,transparent sides at least in a portion and a top face opened; and a capunit which has a second electrode on the outside of a bottom face and isinserted in the base unit from the top face to the middle of the baseunit to be fixed.

Since biopolymer probes are fixed on the first electrode or the secondelectrode, a complementary strand biopolyiner and a non-complementarystrand biopolymer can be separately detected.

Further, the cross section of the pillar-shape base unit is a square andthe cross section of the cap unit is a round shape. Therefore, since alight incident plane is a plane surface, it is possible to suppresslight scattering and easily insert the cap unit into the base unit.

Also, the cartridge further includes a solution reservoir on an upperportion of the base unit for collecting a biopolymer solution overflowedfrom said pillar-shape portion to prevent the solution from flowing outso that it is possible to prevent the solution from flowing out to theoutside.

In the apparatus for detecting of the present invention, a sample DNA isinjected between electrodes facing each other. In this technology, sincean existing amount of DNA can be physically measured, a concentrationthereof and the like can be also determined. Further, by applying anexternal force by an electric field between the facing electrodes toattract single strand probe DNA fixed on the surface of the electrodeand non-hybridized sample DNA to the electrode where the probe DNA isnot fixed, it becomes possible to detect a gene without washing.

Further, by use of this method, clearer results can be obtained sinceboth of reacted one and non-reacted one are targeted for themeasurement.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing a structure of an apparatus fordetecting biopolymers according to one embodiment in the presentinvention.

FIG. 2 is a view showing a structure of a cartridge according to oneembodiment in the present invention.

FIG. 3 is a general view showing an apparatus for detecting biopolymersaccording to one embodiment in the present invention.

FIG. 4 is a schematic view showing a structure of an apparatus fordetecting biopolymers using plate-shape cartridges.

FIGS. 5A and 5B are views showing a plate-shape cartridge in detail.

FIG. 6 is a general view showing an apparatus for detecting biopolymersusing the plate-shape cartridges.

FIG. 7 is a view showing behaviors of DNA when direct current voltage isapplied.

FIG. 8 is a view showing behaviors of DNA when alternating currentvoltage is applied.

DETAILED DESCRIPTION

Hereunder, referring to drawings, preferred embodiments will bedescribed in details.

FIG. 1 is a schematic view showing a structure of an apparatus fordetecting biopolymers according to one embodiment of the presentinvention. The apparatus includes an optical system to measure opticalenergy and the like, such as absorbance, transmittance and reflectance,and an optical system to detect a modified part when DNA is modifiedwith an organic material or an inorganic material, such as a fluorescentmaterial and a radio active material.

The optical system to measure optical energy and the like, such asabsorbance, transmittance and reflectance, includes a laser, an opticalsource, a slit, a filter, a diffraction grating, a light receiving unitand the like. A controller 3 is connected to a computer 1 having adisplay 2. Light generated from a laser and optical source 4 controlledwith the controller 3 is passed through an optical source slit 6 afterwavelength selection with a filter 5. The light is passed through anincidence slit 7 and converted to have wavelengths of 260 nm and 280 nmat a diffraction grating 8. Further, the light is passed through anejection filter 10 placed just before a cartridge 11, the details ofwhich are shown in FIG. 2). Optical energy is decreased in the cartridge11 depending on an existing amount of DNA since DNA absorbs the light.That is, optical energy after the decrease is obtained at a lightreceiving unit 12. Any desired measurement location can be selectedusing an XY stage 13, and reading can be conducted in a scanning manner.Analysis of the result is carried out with the computer 1, and adistribution of the DNA existing amount can be determined by measuringhow much the quantity of optical energy received at the light receivingunit is decreased from incident light at some place. The transmittanceis obtained as a ratio of a quantity of light to that in the case of nopresence of DNA under the condition that the cartridge 11 is fullyfilled with the solution. The reflectance is, in the same manner,obtained as a ratio of a quantity of reflected light to that in the caseof no presence of DNA. In order to measure this reflectance, an opticalsystem to receive the reflected light is needed. The absorbance isobtained by subtracting transmittance from reflectance. Temperatureinside the cartridge can be controlled by a cartridge fixed portion 14provided with an electric heater that is connected to a power source 15.Thus, the temperature during reaction or measurement can be controlled,and the reactivity at each temperature, such as a dissociationtemperature for single strands, for example, can be measured.

The optical system to detect a modified part when DNA is modified withan organic material or an inorganic material, such as a fluorescentmaterial or a radio active material, includes a laser, a light source, apinhole, a lens and the like. Light generated from a laser and lightsource 16 is condensed with a lens 22 after passing through a filter 21for wavelength selection and is passed through a pinhole 23 at the focalpoint. The light passed through the pinhole 23 is again condensed withthe lens 24 having the focal point at a measuring portion. The lightindicating a material excited by the condensed light is advanced to thelens 24 and is advanced through a polarized beam splitter 17 to a lightreceiving unit 18 side. The light having a selected wavelength bypassing through a filter is passed through a pinhole 19 at the focalpoint to reach the light receiving unit 18. A distribution of modifiedparts is analyzed based on signals from the light receiving unit 18.

FIG. 2 is a view showing a structure of a cartridge according to oneembodiment of the present invention. A cartridge 11 includes a cap unitand a base unit 27. In the cap unit 25, at a top face thereof, an innercylinder 25 b having a bottom face opened and a smaller cross section iscoaxially joined to an outside pillar 25 a having a bigger crosssection. An electrode 26 is provided on the outside of the whole bottomface of the inner cylinder 25 b. The base unit 27 has a round shapeelectrode 28 on the inside of the bottom face of a hollow square pillar27 a having a square cross section. A top face of the square pillar 27 ais opened so that a DNA solution is injected and the cap unit 25 can beinserted. Further, a solution reservoir 27 b is provided on an upperportion of the square pillar 27 a to prevent the DNA solution fromflowing out to the outside when some of the DNA solution overflows fromthe square pillar 27 a. With respect to the cartridge 11, there are oneswhere DNA probes are fixed to both electrodes, DNA probes are fixed toone of the electrodes and DNA probes are fixed to neither electrode. Thecartridge 11 is inserted into a cartridge insertion portion of theapparatus. The apparatus has electrodes to generate an electric field inthe cartridge 11 so that direct current voltage and/or alternatingcurrent voltage supplied from a power source can be placed between theelectrodes in the cartridge 11.

Detection is carried out by measuring optical energy such as absorbance,transmittance and reflectance of light having a wavelength of 260 nm.The measurement is carried out by comparison in a plurality of ranges orscanning in a tiny range. Based on a distribution of the obtainedoptical energy such as absorbance, transmittance and reflectance, adistribution of DNA existing between the electrodes can be obtained todetermine an existing amount, a concentration, a hybridization ratio, ahybridization efficiency of DNA and the like.

FIG. 7 is a view showing DNA behaviors when direct current voltage isapplied. When direct current voltage is applied between an electrode 71and an electrode 72, DNA is drawn in a direction of the electric fieldand attracted to one of the electrodes, electrode 72 in this case. Forthis reason, when probe DNA 73 is fixed on the electrode 71 and directcurrent voltage applied between the electrodes after hybridizationreaction, complementary strand sample DNA 75, which was hybridized, isfixed to the electrode 71 to be prolonged, but non-complementary strandsample DNA 76, which was not hybridized, is attracted to the electrode72 side to be a shrunk state. By measuring the DNA amount at eachlocation in this state, the amount and the base length of the hybridizedcomplementary strand sample DNA 75 and the amount of thenon-complementary strand sample DNA 76, which was not hybridized, can bedetermined. Specifically, the base length can be determined by measuringwhere the end of DNA is prolonged and exists.

FIG. 8 is a view showing DNA behaviors when alternating current voltageis applied. When alternating current voltage is applied between anelectrode 77 and an electrode 78, DNA is drawn, in a prolonged state,from the location before the application of voltage to the closerelectrode at some range of frequency and voltage, 1 MHz and 106 V/m inthe present apparatus. In the present embodiment, a sample DNA 79existing at a location closer to the electrode 77 becomes elongated atthe location closer to the electrode 77. A sample DNA 80 existing at alocation closer to the electrode 78 becomes elongated at the locationcloser to the electrode 78. By separately and repeatedly using directcurrent voltage and alternating current voltage as voltage applied tothe cartridge 11, it is possible to control the location of DNA. In thepresent apparatus, direct current voltage is applied between theelectrodes at each of the stage of attracting DNA between electrodes atthe time of injection of a sample, the stage of attracting sample DNA tothe probe side before hybridization reaction and the stage of separatingsample DNA forming a duplex with the probes and non-reacted sample DNAafter hybridization reaction. Alternating current voltage is appliedwhen DNA is stretched in a separated state at the time of measuring thebase length and the like.

FIG. 3 is a general view of the apparatus for detecting biopolymersaccording to one embodiment of the present invention. A rotary cartridgeinserting part 30 is provided in the main unit 29 of the apparatus. Theplurality of cartridges 11 are loaded thereon, and a cover unit 31 ofthe apparatus is closed. Therefore, DNA detection can be continuouslyconducted by automatically changing the cartridges 11 to be subjected tothe measurement.

FIG. 4 is a schematic view showing a structure of the apparatus fordetecting biopolymers using a plate-shape cartridge, the details ofwhich is shown in FIG. 5. An optical system to measure optical energyand the like, such as absorbance, transmittance and reflectance,includes a laser and a light source, a slit, a filter, a diffractiongrating, a light receiving unit and the like. Light generated from alaser and light source 35, after wavelength selection with a filter 36,is passed through a light source slit 37. The light is passed through anincident slit 38 and converted to have wavelengths of 260 nm and 280 nmwith a diffraction grating 39. Further, the light is passed through anejection slit 41 placed just before the cartridge. Optical energy isdecreased in a plate-shape cartridge 42 depending on an existing amountof DNA since DNA absorbs the light. That is, optical energy after thedecrease is obtained at a light receiving unit 45. Any desiredmeasurement location can be selected using an XY stage 43, and readingcan be conducted in a scanning manner. Analysis of the result is carriedout with a computer 32, and a distribution of the DNA existing amountcan be determined by measuring how much the quantity of optical energyreceived at the light receiving unit 45 is decreased from incident lightat some place.

An optical system to detect a modified part when DNA is modified with anorganic material or an inorganic material, such as a fluorescentmaterial or a radio active material, includes a laser and a lightsource, a pinhole, a lens and the like. Light generated from a laser andlight source 47 is condensed with a lens 49 after passing through afilter 48 for wavelength selection and is passed through a pinhole 50 atthe focal point. The light is turned to the plate-shape cartridge 42with a reflecting mirror 51 and is again condensed with the lens 53having the focal point at a measuring portion. The light indicating amaterial excited by the condensed light is advanced to the lens 53 andis advanced through a polarized beam splitter 52 to a light receivingunit 56. The light having a selected wavelength by passing through afilter 54 is passed through a pinhole 55 at the focal point to reach thelight receiving unit 56. Analysis of the distribution of modified partsis conducted with a computer 32 based on signals from the lightreceiving unit 56. Temperature inside the cartridge can be controlled bya cartridge fixed portion 44 provided with an electric heater that isconnected to a power source 46. Thus, the temperature during reaction ormeasurement can be controlled, and the reactivity at each temperature,such as a dissociation temperature for single strands, for example, canbe measured.

FIGS. 5A and 5B are views showing a plate-shape cartridge in details. Aplate-shape cartridge shown in FIG. 5A has a structure, in which storingditches 59 having micro widths and depths are provided on a plate, andelectrodes 57 and 58 are provided on the both sides of each of thestoring ditches 59. When the absorbance, the transmittance and the likeare measured, a transparent bottom face is needed. As for themeasurement, optical energy such as absorbance, transmittance andreflectance and the like is measured. The conventional detection byfluorescence can be carried out.

FIG. 5B is a view showing plate-shape cartridges having gel. By puttinggel 65 in the middle of the storing ditches and heating electrodes 61and 62, it is possible to conduct a time lag measurement. Single-strandDNA probes are fixed to one electrode 61 beforehand and a sample DNAsolution is injected in each of wells 63 located on the side of theelectrode 61, to which the probes are fixed, for hybridization reaction.Measurement is performed for non-reacted sample DNA by conventionalelectrophoresis. When DNA and modified materials in gel portion 65 havecompletely flowed out into the well 64 located on the side of theelectrode 62 facing the electrode 61, the electrode 61 is heated todissociate DNA existing around the electrode 61 to single strands, andthe measurement is again performed by conventional electrophoresis. Bythis way, it is possible to determine a base length distribution and anexisting amount of complementary strand DNA and a base lengthdistribution and an existing amount of non-complementary strand DNA inthe sample DNA.

In the measurement and the detection, it is possible to use unmodifiedsample DNA, but it is possible to obtain higher sensitivity by modifyingDNA with an organic material or an inorganic material, such as afluorescent dyestuff, for excitation from an outside stimulus.

FIG. 6 is a general view of the apparatus of detecting biopolymershaving plate-shape cartridges. A plurality of plate-shape cartridges 42are loaded in a main unit 66 of the apparatus so that the electrodes ofthe cartridge are connected to be in contact with one electrode 67 andthe other electrode 68. The cartridges are scanned in one time with ascanning part 69. Usually, hybridization or electrophoresis is conductedwhile a lid 70 is closed.

Note that the present invention is not limited to the embodimentmentioned above.

In the embodiment mentioned above, the cross section of the cartridge isa square, but other shapes such as a hexagon may be acceptable. It isdesirable that the cartridge has transparent and parallel planes inorder to avoid scattering of light passing therethrough.

Moreover, dissociation temperature for single strands of DNA can bedetermined by varying temperature of the electrodes and by measuring anamount of hybridized DNA or non-hybridized DNA at each temperature.

In accordance with the present invention, the presence and an existingamount or a concentration of a biopolymer such as DNA, RNA and proteinand the like in a sample can be simply determined.

1. A cartridge comprising: a pillar-shape base unit capable ofaccommodating a biopolymer solution, the base unit having a firstelectrode on the inside of a bottom face, transparent sides at least insome portion and a top face opened; and a cap unit which has a secondelectrode on the outside of a bottom face and is inserted to be fixed insaid base unit from the top face to the middle of the base unit.
 2. Thecartridge according to claim 1, wherein a biopolymer probe is fixed onthe first electrode or the second electrode.
 3. The cartridge accordingto claim 1, wherein a cross section of said pillar-shape base unit is asquare and a cross section of said cap unit is a round shape.
 4. Thecartridge according to claim 1, wherein a biopolymer probe is fixed onone of the first electrode and the second electrode, and a cross sectionof said pillar-shape base unit is a square and a cross section of saidcap unit is a round shape.
 5. The cartridge according to claims 1,further comprising a solution reservoir on an upper portion of said baseunit for collecting a biopolymer solution overflowed from saidpillar-shape portion to prevent the solution from flowing out to theoutside.
 6. The cartridge according to claim 1, wherein a biopolymerprobe is fixed on one of the first electrode and the second electrode,and further comprising a solution reservoir on an upper portion of saidbase unit for collecting a biopolymer solution overflowed from saidpillar-shape portion to prevent the solution from flowing out to theoutside.
 7. The cartridge according to claim 1, wherein a cross sectionof said pillar-shape base unit is a square and a cross section of saidcap unit is a round shape, and further comprising a solution reservoiron an upper portion of said base unit for collecting a biopolymersolution overflowed from said pillar-shape portion to prevent thesolution from flowing out to the outside.